Therefore, in the present study, we evaluated the ability of another key amino acid, tryptophan, to enhance the intestinal absorption of biopharmaceuticals. We demonstrated that co-administration with l-tryptophan significantly facilitated the oral and intestinal absorption of the peptide drug insulin administered to rats. Furthermore, l-tryptophan exhibited the ability to greatly enhance the intestinal absorption of other peptide drugs such as glucagon-like peptide-1 (GLP-1), its analog Exendin-4 and macromolecular hydrophilic dextrans with molecular weights ranging from 4000 to 70,000 g/mol. However, no intermolecular interaction between insulin and l-tryptophan was observed and no toxic alterations to epithelial cellular integrity—such as changes to cell membranes, cell viability, or paracellular tight junctions—were found. This suggests that yet to be discovered inherent biological mechanisms are involved in the stimulation of insulin absorption by co-administration with l-tryptophan. These results are the first to demonstrate the significant potential of using the single amino acid l-tryptophan as an effective and versatile bioavailability enhancer for the oral delivery of biopharmaceuticals.Cell-penetrating peptides (CPPs) have great potential to efficiently deliver drug cargos across cell membranes without cytotoxicity. Cationic arginine and hydrophobic tryptophan have been reported to be key component amino acids for cellular internalization of CPPs. We recently found that  During the past few decades, cell-penetrating peptides (CPPs) have been established as potential tools for delivering bioactive macromolecules and lipidic or polymeric particulate carriers into cells via mainly covalent conjugation ,2. Convel-form of the amino acid arginine (l-arginine) could strongly enhance the intestinal absorption of insulin in its single amino acid form, although its effectiveness was relatively less than peptide forms of arginine [l-arginine. However, little is known about the ability of tryptophan to enhance the absorption of insulin.Alternatively, in an earlier study examining the effects of arginine peptides with different chain lengths 4–16 residues), Futaki and coworkers found that the cationic amino acid arginine is a key common amino acid in arginine-rich CPPs, including the human immunodeficiency virus (HIV)-1 Tat peptide and identified an optimal arginine chain length that maximized cellular internalization efficiency . We alsoarginine . Other sarginine ,15,16,17 residuesarginine ,19,20. Iarginine ,6,21,22.Therefore, in the present study, we examined the effects of single amino acid tryptophan on the intestinal absorption of insulin and compared its effectiveness to other useful and conventional absorption enhancers including CPP (penetratin), tight junction-modulator  ,24,25 anl-tryptophan, d-tryptophan, l-phenylalanine, l-isoleucine and l-proline) were purchased from Wako Pure Chemical Industries, Ltd. . Exendin-4 was synthesized by Sigma Genosys, Life Science Division of Sigma-Aldrich Japan Co. . l-Penetratin  and GLP-1 were synthesized by Toray Research Center, Inc. . d-R8  was synthesized by Scrum Inc. . Fluorescein isothiocyanate-labeled dextran with average molecular weights 4400, 20,000 and 70,000 g/mol , sodium taurodeoxycholate and Triton X-100 were purchased from Sigma-Aldrich Co. . C10 was purchased from Tokyo Chemical Industry, Co. Ltd. . Methylcellurose  was purchased from Shin-Etsu Chemical Co., Ltd. . Human colon adenocarcinoma-derived Caco-2 cell line was purchased form the American Type Culture Collection  at passage 18. Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, nonessential amino acids (NEAA), antibiotic mixture , 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) and Hanks’ balanced salt solution (HBSS) were purchased from Gibco Laboratories . Fetal bovine serum (FBS) was purchased from Biowest . Carboxymethyl dextran (CM5)-coated sensor chips were purchased from GE Healthcare . All other chemicals were of analytical grade and are commercially available.Recombinant human insulin (27.5 IU/mg) and amino acids  containing 0.001% MC, which prevents the adsorption of insulin and penetratin to the tube surface and was then normalized with 100 μL of 0.1 M NaOH. The final insulin concentration of the stock solution was 40 IU/mL. Stock solutions of other peptide drugs (GLP-1 and Exendin-4) and macromolecular compounds  were created by dissolving in PBS (pH 7.4) containing 0.001% MC . Two times concentrations of amino acids  were prepared using PBS (pH 7.4) containing 0.001% MC. To aid in the dissolution of high concentrations of hydrophobic amino acids such as l- or d-tryptophan and l-phenylalanine, the solutions were immersed in a water bath at 60 °C resulting in a clear solution. To create the solution used for the intestinal absorption study, two times concentrations of peptide drugs or macromolecular compounds and amino acid stock solutions were mixed together in equal volumes.To prepare the insulin solution, specific amounts of recombinant human insulin were dissolved in 100 μL of 0.1 M HCl. The insulin solution was diluted with 0.8 mL of phosphate buffered saline   containing 0.001% MC at appropriate concentrations. For the in vitro study with Caco-2 cells, HBSS  or completed DMEM (WST-8 assay) were used instead of PBS to produce the test solution.In the comparison study, specific amounts of Animal experiments were performed at Kobe Gakuin University and complied with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals (approved as A16-15 and A17-4). Male Sprague Dawley rats weighing 180–220 g and male ddY mice weighing 30–40 g were purchased from Japan SLC, Inc. , housed in temperature controlled rooms (23 ± 1 °C) with a relative humidity of 55 ± 5% and had free access to water and food during acclimatization. Animals were fasted for 18 h (rats) and 24 h (mice) before the experiments; however, they were allowed to drink water ad libitum.®, Kyoritsu Seiyaku Corp., Tokyo, Japan), which has been conventionally used in our insulin delivery study as confirmed to have a minimal effect on the blood glucose levels, rats were restrained in a supine position on a thermostatically controlled board at 37 °C. Additional i.p. injections of sodium pentobarbital (12.5 mg/kg) were administered every 1 h to maintain anesthesia. The ileum was exposed following a small midline incision made carefully in the abdomen. Its proximal to ileocecal junction segments (length = 10 cm) were cannulated at both ends using polypropylene tubing and ligated securely to prevent fluid loss. To wash out the intestinal contents, PBS warmed to 37 °C was circulated through the cannula at 5.0 mL/min for 4 min using an infusion pump. The cannulation tubing was removed, the segments were closed tightly and returned carefully to their original location inside the peritoneal cavity. Rats were kept on the board at 37 °C for an additional 30 min to calm biological reactions such as elevated blood glucose levels resulting from the stress imposed by surgery. After 30 min rest period, 0.5 mL of test insulin or other drug solution with or without amino acids and/or l-penetratin was administered directly into a 6 cm ileal loop created from the 10 cm pretreated segment. The insulin dose was administered at 50 IU/kg body weight (20 IU/mL) for all animal experiments. To examine dose dependency, the final concentrations of tryptophan were adjusted to 4, 8, 16 and 32 mM. In the comparison study with three hydrophobic amino acids or metabolite of l-tryptophan, the final concentrations of l-isoleucine, l-proline, l-phenylalanine and serotonin were 32 mM. In the study examining the synergistic effect of l-arginine and l-tryptophan, the final concentration of l-arginine was fixed at 40 mM. The doses of GLP-1 and Exendin-4 were both 1.25 mg/kg (0.5 mg/mL) and the doses of FDs were all 10 mg/kg (4 mg/mL). In the study examining the effect of pretreatment with l-tryptophan, d-R8, l-penetratin and other positive controls, ileal loop was exposed to these solutions during 30 min rest period after surgery and before the administration of insulin solution. For a part of above experiments involving pretreatment with l-tryptophan, C10 and sodium taurodeoxycholate for 30 min, an additional rest period of 30 min was added before the administration of insulin.Following anesthetization by intraperitoneal (i.p.) injection of sodium pentobarbital , GLP-1 ELISA kit  and Exendin-4 EIA kit , respectively. The plasma concentrations of FDs were measured with a microplate fluorimeter  at excitation and emission wavelengths of 485 and 528 nm, respectively. The total area under the drug concentration time curve (AUC) from 0–3 h was estimated using the sum of successive trapezoids fitted between each set of data points in the time profiles of the plasma drug concentration.During the experiment, 0.25 mL blood aliquots were taken from the jugular vein at 0, 5, 10, 15, 30, 60, 120 and 180 min after dosing. The tuberculin syringes  were heparinized by the standard procedure of coating the syringe wall with aspirated heparin and then expelling all the heparin. Plasma was separated by centrifugation at 13,400× Cmax) and time to reach Cmax (Tmax) were determined directly from the plasma insulin concentration–time curves. The total area under the insulin concentration–time curve (AUC) for 0–3 h was estimated from the sum of successive trapezoids between each data point. The relative bioavailability (BA) of insulin was calculated relative to the s.c. injection as follows:The bioavailability of intestinal administered insulin was calculated relative to the subcutaneous (s.c.) route. Briefly, an insulin solution was prepared by dissolving an appropriate amount of insulin in PBS for s.c. injection (1 IU/kg). The peak plasma concentration (l-tryptophan (32 mM) (100 μL) by using oral gavage tube (i.d. 0.9 × length 50 mm) . Blood glucose levels were measured  by collection of blood (one drop) from a small cut in the tail vein at t = 0 (prior to dosing), 15, 30, 60, 120, 180, 240, 300 and 360 min after administration. Blood glucose levels are depicted as percentage of the initial value in same mouse. Mice were trained with oral dosing and tail cutting at 24 h and 60 min prior to actual drug administration to avoid the unexpected elevation of blood glucose levels during experiments.After 24 h fasting, mice were dosed with insulin (50 IU/kg) with or without l- or d-tryptophan  was mixed with insulin  and incubated in the intestinal fluid at 37 °C. At 5, 15, 30, 45 and 60 min, 50 μL was collected and added to 50 μL of ice-cold mobile phase solution to terminate the reaction. l- or d-penetratin  and STI  were used as a CPP and positive conventional inhibitor, respectively. Insulin concentration was measured by HPLC  using the following conditions: mobile phase, acetonitrile, trifluoroacetic acid (0.1%) and sodium chloride ; 20 μL injection volume; 1.0 mL/min flow rate; 220 nm wave length; analytical column  with guard column  .Intestinal fluid was collected from male Sprague-Dawley rats by inserting a Sonde needle into the upper portion of the small intestine and the intestine was then cannulated on the lower side (length = 20 cm) to remove the intestinal fluid. The contents of the small intestine were washed out with 30 mL of PBS. Because intestinal fluid contains a high lipid content, the efflux solution was treated with equal volume of methylene chloride to remove any lipids that might interfere with the analysis of insulin by HPLC. This removal of lipid contents was repeated five times. 2 culture dishes  with 10 mL of culture medium. The culture medium consisted of DMEM containing 10% FBS, 0.1 mM NEAA, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (completed DMEM). The seeding density for cultivation was 8.0 × 105 cells/dish. The cells were maintained in an incubator at 37 °C, 95% relative humidity and 5% CO2. The culture medium was replaced with fresh medium every second day for approximately 7 days until the cells reached 80% confluence, at which time they were subcultured. For the subculture procedure, the cells were detached from the culture dishes by trypsinization with 0.05% trypsin–EDTA, counted with a hemocytometer and transferred at the desired seeding density to new culture dishes or experimental wells. We used cells between passages 25 and 35.Caco-2 cells were cultured in 75 cml-tryptophan, the integrity of the cell membrane after treatment with the CPPs was examined by measuring the lactate dehydrogenase (LDH) released from the cytoplasm under both in vitro and in situ conditions.To assess the cytotoxic effects of 2 culture area (Nippon Becton Dickinson) at the density of 1.0 × 105 cells/cm2 and then grown in completed DMEM for 3 or 4 days. Before the addition of l-tryptophan, cell membranes were allowed to equilibrate in 0.5 mL of assay buffer for 30 min. HBSS containing 0.001% MC (pH 7.4) was added to the wells as the transport buffer. After a 30 min pre-incubation with transport buffer, the assay was initiated by replacing 50 μL of transport buffer with 50 μL of l-tryptophan stock solution (ten times concentration) or transport buffer. Various concentrations (300–2400 μM) of l-tryptophan were incubated in Caco-2-seeded wells for 120 min at 37 °C. The incubation buffer was collected and LDH released from the cytoplasm into the incubation buffer was determined using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit . Cytotoxicity was expressed as the percentage calculated by dividing the absorbance of the vehicle (HBSS) or the l-tryptophan-treated sample by that of a sample treated with 0.8% Triton X-100, according to the instructions of assay kit.In the in vitro assay, the Caco-2 cells were grown in 24-well plates with a 1.88 cml-tryptophan solution  with 50 IU/kg insulin were administered into rat ileal segments in the same manner as the closed loop administration study described above. For the trial with the highest concentration of l-tryptophan (50 mM), l-arginine (40 mM) was added as a solubilizing agent. For this test, 5% sodium taurodeoxycholate was used as a positive control. At 60 min after administration, the ileal loop was washed with 5.0 mL of PBS and then intestinal fluid was collected. After adjusting the collected intestinal fluid to a total volume of 6.0 mL with PBS, the activity of LDH (Unit) leaked into the intestinal fluid was determined using an LDH kit (Promega Corp.).In the in situ assay, various concentrations of l-tryptophan, a WST-8 assay was performed with Cell Counting Kit-8 . The Caco-2 cells were grown in 96-well plates with a 0.33 cm2 culture area (Nippon Becton Dickinson) to a density of 3.0 × 104 cells/cm2 and then grown in 100 μL of completed DMEM for 3 or 4 days. In this assay, the completed DMEM was used in place of the incubation buffer to prevent the possible loss of cell viability during total 5 h incubation in HBSS. First, 10 μL of l-tryptophan stock solution (eleven times concentration) or transport buffer was added to 100 μL of completed DMEM and the cells were incubated with various concentrations of l-tryptophan (600–2400 μM) for 2 h. Then, 10 μL of CCK-8 reagent was added to the wells and the cells were incubated for an additional 3 h to allow for the reductive reaction of WST-8. The resulting absorbances of the incubated solution in the wells were read with a microplate reader  at a wavelength of 450 nm. In this assay, 0.8% of Triton X-100 was used as a positive control, same as the LDH assay.To examine the cellular viability of Caco-2 cells exposed to 2 culture area (collagen-coated polytetrafluoroethylene (PTFE) membrane with 0.4 μm pore size, Corning Inc., Corning, NY, USA). The cells were cultured to a density of 1.0 × 105 cells/cm2. The cells were grown in completed DMEM for 21 days until they achieved a constant TEER reading of >500 Ω cm2, which indicated that tight junctions had formed in the monolayer. The culture medium was changed every second day and the electrical resistance was monitored by the method described below. Each well consisted of apical (top) and basal (bottom) chambers, which were separated by a collagen-coated polytetrafluoroethylene membrane with a pore size of 0.4 μm and a filter area of 1.12 cm2.For TEER measurement, Caco-2 cells were grown in 12-well transwell plates with a 1.13 cml-tryptophan or serotonin stock solutions (ten times concentration) or transport buffer. l-tryptophan  or serotonin (2.4 or 16 mM) was incubated with insulin (15 μM) in the apical chamber of the Caco-2-seeded transwell for 2 h. During the incubation, the Caco-2 monolayer was kept at physiological temperature (37 °C).In preparation for the assays, the cell membranes were allowed to equilibrate for 30 min in transport buffer; HBSS (pH 7.4) with 0.001% MC was added to the apical (0.5 mL) and basal (1.5 mL) chambers of the transwell. After a 30 min pre-incubation for equilibration, an initial TEER measurement was taken of the monolayer in each well and then 100 μL of transport buffer was removed from the apical chamber and replaced with 50 μL of insulin and 50 μL of l-tryptophan on the intercellular tight junctions. The TEER is expressed as a percentage calculated by dividing the TEER value (Ω cm2) at 2 h by the initial value.The TEER of the Caco-2 monolayer was measured to confirm cell growth and differentiation on the insert filter of the transwell using a Millicell ERS-2 . Before seeding the Caco-2 cells, the electrical resistance of the insert filter in the medium was measured and subtracted from the total electrical resistance determined for the monolayer to calculate the intrinsic TEER of the monolayer. The TEER of the monolayer was measured at the end of the 2 h incubation to detect any negative effects of the applied l-tryptophan or l-penetratin were analyzed by SPR . To measure the binding of l-tryptophan or l-penetratin to insulin, insulin was immobilized at the carboxymethyl dextran surface of a CM5 sensor chip by using an amine coupling method. For the immobilization procedure, insulin was diluted to a concentration of 50 μg/mL using acetate buffer at pH 4.5 and immobilized on the chip surface in separate flow cells at 5 μL/min for 7 min. Reference surfaces were prepared by amine coupling activation followed by immediate deactivation. For binding measurements, different concentrations of l-tryptophan or l-penetratin (2–200 μM) were injected for 90 s followed by an additional 90 s dissociation phase. At the end of each cycle, the surface was regenerated by a 30 s injection of 1 M NaCl. Measurements were carried out in HBSS (pH 7.4) supplied with 0.001% MC at 20 μL/min at 25 °C. Each sensorgram was determined by subtracting nonspecific binding on the surface of the reference flow cell from total binding on the immobilized-insulin surface.The intermolecular interactions between insulin and p value was less than 0.05.Each value is expressed as the mean and standard error of the mean (SEM) of multiple determinations. The significance of the differences in the mean values of multiple groups was evaluated using analysis of variance with Dunnett’s test. IBM SPSS Statistics  was used for statistical analysis. Differences were considered significant when the l-form of tryptophan (8–32 mM) into rat ileal closed loops. As shown in l-tryptophan in a concentration dependent manner and with a late onset of action 30–60 min after administration. This result suggests that l-tryptophan has the potential to strongly enhance the intestinal absorption of insulin as single amino acid form. On the other hand, we confirmed the effect of the amphipathic CPP penetratin on the intestinal absorption of insulin. The effect of l-penetraitn shown in l-penetratin [l-penetratin was absorbed immediately at 15 min post administration. The difference of action onset between l-tryptophan and l-penetratin suggested that l-tryptophan as single amino acid played a role in increasing absorption of insulin in a different way from tryptophan residues as a component of the peptide structure of l-penetratin. We further compared the effect of other hydrophobic amino acids such as phenylalanine, proline and isoleucine on the absorption of insulin. The results in It was reported that tryptophan residues at position 6 and 14 in the penetratin amino acid sequence aid in lipid interaction in cell membranes and are therefore an important moiety involved in the cellular internalization of penetratin . To testtophan 8–2 mM intol-tryptophan has the ability to effectively increase the intestinal absorption of insulin. Therefore, the potential of l-tryptophan as oral absorption enhancer was further tested after in vivo oral administration of insulin to mice. As shown in l-tryptophan (32 mM) suggested that l-tryptophan could enhance the oral absorption of insulin. The elevation in the blood glucose levels just after oral administration in all groups might be because of the stress by oral gavage, even though mice were trained before administration.The above-mentioned results suggested that l-tryptophan is still unclear, our results strongly suggest that l-tryptophan has potential as an effective absorption enhancer for the oral delivery of insulin. We additionally researched the ability of l-tryptophan to aid in the delivery of other biopharmaceuticals.Although the mechanism involved in the absorption-stimulatory effect of l-tryptophan to assist in the transport of a range of differently sized potential drug cargo, we evaluated the effect of l-tryptophan co-administered with fluorescently labeled hydrophilic dextrans of various molecular weights . As shown in l-tryptophan (32 mM), although the effectiveness of enhancement tended to decrease with increase in the molecular weight of the cargo compounds. Furthermore, the effect of l-tryptophan on the intestinal absorption of other peptide drugs, GLP-1 and Exendin-4, was examined. As shown in l-tryptophan (32 mM). Thus, l-tryptophan has the potential to enhance the intestinal absorption of a wide variety of biopharmaceuticals in a safe manner. The AUC values of dextrans and peptide drugs are summarized in To examine the ability of l-tryptophan might be attributed to the modulation of biological structures and functions in the intestinal mucosa. To establish the safety of l-tryptophan as a potential absorption enhancer for biopharmaceuticals, changes in the integrity of epithelial cell membranes, cellular viability and the tightness of the paracellular junctions after administration of l-tryptophan were examined.The intestinal absorption of insulin accelerated by co-administration with l-tryptophan at all tested concentrations (16 and 32 mM) and these measurements were significantly low when compared to the positive control (the administration of 5% sodium taurodeoxycholate). Similarly, in the in vitro assay . l-tryptophan to culture media. The results indicate no decrease in cell viability after addition of l-tryptophan at all concentrations (600–2400 μM). The toxicity examinations suggest that l-tryptophan has no adverse effect on the intestinal mucosa and may therefore become a potentially safe absorption enhancer.ro assay B, no rell-tryptophan can enhance the intestinal absorption of insulin via functional and structural modification of the intestinal mucosa, while not exhibiting cytotoxicity , CPPs (d-R8 and l-penetratin), or conventional enhancers (C10 and sodium taurodeoxycholate) for 30 min and then washing the pretreated ileal segment with fresh PBS.The results from the previous section suggest that toxicity . When ful-tryptophan and l-penetratin, however, the extent of insulin absorption was lower than the effect of co-administration with these enhancers  or l-penetratin (2–200 μM) were injected into the flow cells containing immobilized insulin. The resulting sensorgrams do not show an increase after the injection of l-tryptophan at any concentration , although a significant reduction in TEER was observed after addition of typical enhancer C10. The maintained TEER confirmed that the cellular lipid membrane was retained after addition of l-tryptophan, consistent with the LDH release assays  and compared its effectiveness to that of l-tryptophan (d-tryptophan appears much weaker than that of l-tryptophan but the onset of action by d-tryptophan (60 min after administration) was similar to that of l-tryptophan.The lack of rt assay B contrad insulin , suggest insulin . As it iyptophan A. The rel-tryptophan, serotonin, has the potential to enhance the epithelial permeability of insulin and other macromolecular drugs [l-tryptophan might be attributed to the time required for conversion from tryptophan to serotonin after administration to the intestine. Therefore, we then tested the effect of serotonin on the permeation and absorption of insulin under the in vitro and in situ conditions. The result of the permeation study with Caco-2 cell monolayer  and l-penetratin (rapid) shown in l-tryptophan  in the in situ ileal loop administration study.The difference in the action onset between yptophan A,B confil-tryptophan (16 mM) and l-penetratin (0.5 mM) on the absorption of FD-4 was confirmed as being delayed and rapid onset, respectively, consistent with the above-mentioned results with insulin (l-tryptophan (16 mM) and l-penetratin (0.5 mM) on the absorption of FD-4 was almost equal to that co-administered with only l-penetratin (0.5 mM). As l-penetratin had the capacity to interact with FD-4 similar to insulin, l-tryptophan might be deprived of a chance to facilitate the absorption of FD-4. Possibly, a large part of FD-4 administered to the intestine could be systemically and quickly absorbed by complexing with l-penetratin, while FD-4 which could be delivered by l-tryptophan was scarce due to the slow mechanism of action. The results suggested that the action mechanism of l-tryptophan to facilitate the intestinal absorption of macromolecular drugs was different from that of l-penetratin.As shown in  insulin . Contrarl-form of arginine, an important component in cationic CPPs, has the potential to facilitate the intestinal absorption of insulin without being part of a peptide structure such as arginine-rich CPPs [We recently discovered that single amino acid arginine, particularly the ich CPPs . Anotherich CPPs ,18. In fich CPPs . Therefol-tryptophan . Interestingly, the onset patterns of insulin absorption after co-administration were quite different between l-arginine (Tmax 12.8 ± 0.9 min at 40 mM) and l-tryptophan (Tmax 52.5 ± 7.5 min at 32 mM), as shown in l-arginine and l-tryptophan, observed in the present and current studies [l-tryptophan and l-arginine could enhance intestinal absorption via mechanisms distinctly different from that involving CPPs.The data showed that the intestinal absorption of insulin was significantly enhanced by co-administration with  studies . This sul-tryptophan . This observation is quite different from the mechanism of penetratin and R8 in which intermolecular interaction is essential to their ability as absorption enhancers. There was no protective effect of l-tryptophan on the enzymatic degradation of insulin  to the transwell with Caco-2 cell monolayers [l-tryptophan (1 mM or more) increases the paracellular permeability of macromolecules in an energy- and sodium-dependent manner [l-tryptophan   A. Theref4 receptor, possibly contributing to the increased intestinal absorption of macromolecules [l-tryptophan  and the action onset of serotonin was delayed compared to l-tryptophan. Furthermore, our in vitro data partially negated the possible cholinergic contribution, as serotonin appeared to tighten the paracellular space and decrease the epithelial transport of insulin through a Caco-2 cell monolayer , it was considerably small compared to that of l-tryptophan . In this study, insulin and l-tryptophan were administered just as a solution, therefore, the development of an appropriate formulation could be expected to increase the biological effect of insulin. l-tryptophan as an absorption enhancer for macromolecules having peptide- to protein-sized molecular weights. In contrast, l-arginine had no effect on the intestinal absorption of such peptide drugs (data not shown). The effectiveness of absorption enhancement by l-tryptophan was comparable to that by l-penetratin, although l-tryptophan required relatively higher concentrations (16 mM or more) than l-penetratin (0.5 mM). Since l-tryptophan is currently used as an oral supplement and it appears to facilitate the intestinal absorption of a wide variety of biopharmaceuticals, its potential as an intestinal absorption enhancer is meritorious and quite unique.Based on the results of the in situ intestinal insulin absorption study, we examined the potential of l-tryptophan is one of the essential amino acids, its excess use has to be carefully considered for safe pharmacotherapy. In addition, another metabolite from tryptophan, melatonin, is used as a sleep-inducing agent and therefore may affect circadian rhythm. The required dose of l-tryptophan for inducing sleepiness is roughly to 1500–3000 mg·day−1·human−1, which is comparable to the maximum dose used to enhance the intestinal absorption of biopharmaceuticals  in this study. However, the dose of l-tryptophan used as an absorption enhancer could be further optimized by precisely evaluating the relation between its co-administered concentration and the efficiency of the resultant absorption enhancement. Considering the effective plasma concentration of insulin (<100 μU/mL), it is expected that the dose of l-tryptophan can be reduced to 10 mM, the equivalent to 5.1 mg/kg (300 mg/human). Further examination will be essential to establish the potential of l-tryptophan as a safe and effective absorption enhancer for the oral delivery of biopharmaceuticals.As mentioned above, tryptophan is first metabolized into serotonin which stimulates the cholinergic nervous system, balancing neural activity. Therefore, although l-tryptophan was far stronger than that of l-arginine [l-penetratin. However, the mechanism of action remains unclear. In the present study, the cell integrity of the intestinal epithelium  was not changed in the presence of l-tryptophan. Furthermore, no intermolecular interaction between insulin and l-tryptophan was observed and insulin was not protected from enzymatic degradation when in the presence of l-tryptophan. Tryptophan is the precursor of a wide array of bioactive compounds, such as serotonin, melatonin and so forth. [l-tryptophan as an absorption enhancer relative to the d-form, inherent biological functions might be involved in its action mechanisms. To utilize l-tryptophan as a safe absorption enhancer, further studies are required to identify the complete mechanism of action.In the process of studying the role of the hydrophobic amino acid tryptophan, which was widely known as a key amino acid in the function of CPPs, we discovered that tryptophan singly improves the intestinal absorption of various biopharmaceuticals having peptide to protein molecular sizes. When examining the absorption of insulin in particular, the bioavailability enhancement effect of arginine , anothero forth. . Conside


Alpha-fetoprotein-producing gastric cancer (AFP-GC) is a relatively rare disease, with a dismal prognosis.We report the case of a patient with long-term survival after surgery for the recurrence of AFP-GC. A 71-year-old man was diagnosed with gastric cancer and underwent distal gastrectomy with D2 lymphadenectomy (pT3N2M0). Pathological examination of the resected specimen revealed AFP-GC. Fifteen years after the gastrectomy, the patient experienced anorexia and was admitted with a mass located at the mesentery of the small intestine. Following a diagnosis of gastrointestinal stromal tumor of the mesentery, a tumor resection with partial small intestine was performed. The final histopathological diagnosis was AFP-GC’s recurrence in the small-bowel mesentery. Two months later, multiple liver metastases were identified, and serum AFP level was found to be extremely high . Chemotherapy with S-1+CDDP (SP) was initiated for liver metastasis. However, owing to anorexia and fatigue, SP therapy was discontinued following the patient’s request at the end of two courses. A CT scan at 1 month after the discontinuation of chemotherapy did not reveal liver metastasis, and serum AFP level decreased to the normal range. He is alive at present with no re-recurrence and no elevation of serum AFP level at 7 years after the second surgery without any chemotherapy.Even if recurrence of AFP-GC is diagnosed, radical resection and chemotherapy are effective, as noted in the present case. Alpha-fetoprotein-producing gastric cancer (AFP-GC) is a relatively rare disease, accounting for 1.3–15% cases of gastric cancer . AFP-GC Herein, we present a case of a patient with long-term survival after successful treatment with radical surgery for the solitary metastasis of the mesentery and chemotherapy for the multiple liver metastases of AFP-GC at 22 years after the first gastrectomy.A 71-year-old man was admitted to our hospital with complaints of persistent anorexia. Fifteen years ago, he had undergone distal gastrectomy with D2 lymphadenectomy, followed by Billroth-II type reconstruction for gastric cancer. Pathological examination of the gastric tumor revealed an AFP-GC, and the pathological stage was pT3 (SS) N2 M0, stage IIB (Japanese classification of gastric carcinoma: 3rd English edition) [Two months after the second surgery, CE-CT scan revealed multiple liver metastases Fig.  and seruPrevious studies have reported a poor prognosis for AFP-GC with lymphatic and venous invasion, along with high rates of liver metastasis as compared with non-AFP-GC , 8, 9. IOur case highlights three key learning points: first, the long-term  dormancy following the initial gastrectomy until the identification of the solitary metastasis to the mesentery; second, the multiple liver metastases, which went unobserved for 15 years, were evident only 2 months after the second surgery; and last, the disappearance of liver metastases for 5 years following only two courses of chemotherapy with S-1+CDDP.Gastric cancer usually recurs within the first 2 years of gastrectomy. Late recurrences are rare, with approximately < 1% cases recurring after 10 years . PrognosIn an earlier study, Adachi et al. analyzed 270 AFP-GC cases. The authors reported that the 5-year survival rate and median survival period in all patients were 22% and 14 months, respectively. However, they found that patients with curative gastrectomy comprised 42% of the study cohort and median survival period was 29 months . Our patIn Japan, SP therapy for unresectable and recurrent gastric cancers has been recognized as the standard therapy . At the Even if recurrence of AFP-GC is diagnosed, there are cases in which radical resection and chemotherapy are effective, as noted in the present case. Additional accumulation of AFP-GC cases is necessary to identify and select such cases.


We observed no statistically significant increase in the micronucleus (MN) frequency or the level of DNA strand breaks in cells exposed to 40-GHz millimeter waves relative to sham-exposed and incubator controls. Heat shock protein (Hsp) expression also exhibited no statistically significant response to the 40-GHz exposure. These results indicate that exposure to 40 GHz millimeter waves under these conditions has little or no effect on MN formation, DNA strand breaks, or Hsp expression in HCE-T or SRA01/04 cells.Millimeter waves are used in various fields, and the risks of this wavelength range for human health must be carefully evaluated. In this study, we investigated the effects of millimeter waves on genotoxicity and heat shock protein expression in human corneal epithelial (HCE-T) and human lens epithelial (SRA01/04) cells. We exposed the cells to 40-GHz millimeter waves at 1 mW/cm Hence, we sought to investigate the effects of millimeter waves on human epithelial cells. Previously, we studied the effect of long-term exposure to 60-GHz millimeter-wave radiation on micronucleus (MN) formation, DNA strand breaks, and heat shock protein (Hsp) expression in cells derived from the human eye . We sougc fields . On the c fields –12. Thusc fields , 14. To An applicator based on a printed circuit board (PCB) was used to expose cells to 40-GHz millimeter waves. The concept of the applicator has been described in detail in another paper , in whic2 at a relative humidity of >95% and a temperature of 37°C. The applicators for exposure (right) and for sham-exposure (left) were equipped in the incubator. Figure Figure The applicator was made using a PCB with a disc-shaped area surrounded by a post-wall waveguide to feed millimeter waves into the substrate of the disc-shaped area, on which a culture dish could be placed. A culture dish 100 mm in diameter was positioned on the disc-shaped area. The 40-GHz millimeter waves were applied from the bottom through coupling windows of fine slots opened in the top metal layer of the PCB. Thus, adherent cells on the bottom of the culture dish were exposed to millimeter waves. Temperature elevation was suppressed to <0.1°C using a heat sink unit located beneath the PCB.2 at the bottom of culture medium. The distribution of the power density was estimated numerically, and 61% and 82% of the area of the bottom layer of the culture dish was exposed at the power density within an average ±3 dB and ±5 dB, respectively [The spatially averaged power density was set at 1 mW/cmectively .2 in the medium. As positive controls, the cells were treated with 10 μg/ml bleomycin  for 1 h (for the genotoxicity test) or heat treatment (for the Hsp expression test).HCE-T cells  . In a total of 300 binucleated cells, MN formation was counted according to modified criteria described previously [The methodology for the micronucleus (MN) formation test was described previously . Brieflyeviously , using aThe comet assay was performed to detect DNA strand breaks at the single-cell level; the methodology was described previously . Brieflyg for 15 min. Supernatants were collected, and the concentrations were measured using a calibration curve on an iMark plate reader ; protein concentrations were adjusted to 1 mg/ml in all samples. The samples were then mixed with 2ME sample buffer  at a ratio of 1:1, incubated at 100°C for 1 min, and immediately placed on ice. Twenty micrograms of extracted protein was loaded onto 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel , separated by electrophoresis, and transferred to nitrocellulose membrane  by iBlot (Life Technologies Japan). All samples  in three experiments were loaded on one gel. After blotting, the BenchPro™ 4100  was used for the blocking and immunostaining. Membranes were blocked with 1.5% skim milk  for 1 h, and then immunostained with antibodies for 1 h. Primary antibodies were obtained from the indicated suppliers: Hsp27 , Hsp70 , Hsp90α (StressMarq) and β-actin (Sigma-Aldrich). Secondary antibodies were anti-mouse , anti-goat (R&D), and anti-rabbit (Sigma-Aldrich). After immunostaining, membranes were stained with horseradish peroxidase, and then analyzed using the ATTO Image Analysis Software .The methodology for measuring Hsp expression was described previously . BrieflyP-value of <0.05 or <0.01 was considered to be statistically significant.The data were analyzed using Dunnett’s test. A The MN frequencies in HCE-T and SRA01/04 cells are shown in Fig. Two photographs in Fig. The expression levels of Hsp27, 70 and 90α in HCE-T cells are shown in Fig. 2, on cellular genotoxicity and stress responses in HCE-T and SRA01/04 cells [2 for 24 h under the same conditions. The results for each parameter were almost the same as in the previous study.Previously, we investigated the effects of exposure to 60-GHz millimeter-wave radiation, at a constant temperature (37°C) for 24 h at the average power density of 1 mW/cmet al. recently reported that 60.4-GHz millimeter-wave exposure has no effect on protein expression of Hsp70 or certain membrane receptors [et al. also reported that 24-h exposure to 60-GHz millimeter waves has no impact on dopamine turnover or dopamine transporter protein expression in NGF-treated PC12 cells, although they did observe a slight (but insignificant) increase in the level of 3,4-dihydroxyphenylacetic acid [et al. reported no non-thermal effects following exposure of human skin fibroblast and glioblastoma cells to 70–300 GHz using a tunable system [Over the past decade, millimeter-wave radiation has spread widely due to the development of technologies that use these wavelengths. However, the biological effects of millimeter-wave exposure have not been well studied. To date, several studies have examined the biological effects of millimeter-wave exposure on neural activity , cell preceptors . Althougeceptors . Other stic acid , althouge system . In summet al. indicated that 32.9–39.6-GHz exposure enhanced the neutrophil response to particulate agonists [in vivo experiments [In this study, we investigated the genotoxicity of 40-GHz millimeter waves in human eye epithelial cells. Very few studies have examined the effect of this wavelength on this type of cells, but some positive data have been obtained in cells exposed to similar frequencies. Recently, Vlasova agonists . The freeriments –32. HoweThe results of this study suggest that exposure of eye epithelial cells to 40-GHz millimeter-wave radiation has little or no effect on genotoxicity or protein expression. These results were consistent with our previous data obtained with 60-GHz millimeter irradiation. Although several previous studies reported some effects of these wavelengths, the mechanisms remain unclear. In future work, we should perform further experiments for cells derived from other organs, and we will proceed to clarify the effects of millimeter waves using other, highly sensitive detectors, i.e. RNA-sequencing, time-lapse microscopy, etc., because slight biological changes that cannot be detected by existing technologies might be expressed by the millimeter exposure.


Atrial fibrillation (AF), an important cause of thromboembolic events, is often silent and intermittent, thus presenting a diagnostic challenge. The aim of this study was to assess whether the plasma level of N-terminal pro-brain natriuretic peptide (NT-proBNP) is related to the presence of AF and thereby might be used to facilitate screening programs for AF in primary care.This was a cross sectional screening study. A population of 70–74-year-old individuals registered at a single primary care center in Stockholm were invited to AF screening. Intermittent ECG recording, 30 seconds twice a day using a hand-held device over 2 weeks, was offered to participants without previously known AF. Of the 324 participating persons, 34 patients had already known AF and 16 new cases of AF were detected by screening. Plasma NT-proBNP was measured in patients with previously known AF, newly detected AF, and 53 control participants without AF.The median NT-proBNP was 697 ng/L in patients with previously known AF, 335 ng/L in new cases of AF, and 146 ng/L in patients without AF. After adjustment for several clinical variables and morbidities, the differences of median NT-proBNP levels were statistically significant between cases of previously known AF and new cases of AF as well as between new cases of AF and those without AF. The area under receiver operating characteristic curve of detection of new AF was 0.68 (95% CI 0.56 to 0.79) yielding a cut-off point of 124 ng/L with 75% sensitivity, 45% specificity, and 86% negative predictive value.The NT-proBNP plasma levels among patients with known AF are higher than those with newly detected AF, and the latter have higher levels than those without AF. Therefore NT-proBNP might be a useful screening marker for the detection of AF and its persistence. Atrial fibrillation (AF) is a common cardiac arrhythmia carrying a high risk for ischemic stroke . Oral anOpportunistic screening for AF using pulse–palpation is recommended in persons above the age of 65 according to the European Society of Cardiology guidelines . By singScreening for AF among individuals aged 70–74 years in primary care using intermittent electrocardiogram (ECG) recordings for 2 weeks yielded 5.5% individuals with newly detected AF .N-terminal pro B-type natriuretic peptide (NT-proBNP) can be used as a biomarker for predicting the development of AF ,5,6, strThe role of NT-proBNP in screening for AF in primary care has not been studied. The aim of this study was to evaluate the usefulness of NT-proBNP in systemic screening for AF in primary care.https://www.protocols.io/view/the-feasibility-and-outcome-of-atrial-fibrillation-m2fc8bn.3. Briefly, the target population of the study was 415 individuals 70–74 years old who were registered at a single primary care center (PCC). Patients with previously known AF were invited for routine physician visits in the PCC, and individuals without previously known AF and who visited the PCC for consultations for any reason during the one-year inclusion period were also invited to participate in the screening program. The remaining individuals who did not visit the PCC during the inclusion period received a written invitation to participate.The study population was selected from a previous cross sectional screening study for AF , and theParticipants received written and oral information about the study, and they gave their informed and written consent to participate. The responsible physician took the participants’ medical histories, including their current medications, and performed a general medical examination that included blood pressure measurement and fasting plasma glucose. Participants without previously known AF were examined with a 12-lead ECG. When the ECG did not show AF, intermittent handheld ECG (Zenicor) recordings were made for 30 seconds twice a day, and in case of palpitations recordings were made for at least two weeks. When handheld ECG findings showed AF or any other suspected pathological finding, the ECG was re-examined by an experienced cardiologist in order to confirm the diagnosis. Participants with unclear or uninterpretable ECG were further investigated with Holter ECG recordings. Oral anticoagulant treatment was offered to patients with newly detected AF.Plasma NT-proBNP levels were evaluated at the end of the screening study. NT-proBNP was analyzed for all new cases of AF and all known cases of AF. Of those individuals with no detected AF, a non-randomly selected group of 53 individuals (18%) were also checked for NT-proBNP through venous blood sampling . Plasma Categorical data were summarized by counts and percentages. For all continuous variables, visual inspection of histograms and the Shapiro–Wilk’s test were used to assess the deviation from a normal distribution. Normally distributed continuous data are reported as means with standard deviations, whereas non-normally distributed data are reported as medians with interquartile ranges. The CHA2DS2-VASc score  was regaMultivariable analysis was performed using binary logistic regression to test the association between AF and risk factors. The results are presented as odds ratios with 95% confidence intervals. NT-proBNP data were not normally distributed and were therefore normalized using natural logarithmic transformation, and the logarithmic mean was used in the regression test.Receiver operating characteristic analysis was performed, and the discriminative ability was assessed by the area under curve (AUC). Youden’s index was calculated to determine whether it could be used to find a clinically relevant cut-off for NT-proBNP. The specificity balanced with the desired high sensitivity for different cut-offs was then studied to determine a clinically relevant cut-off value.For all tests, a p-value < 0.05 was considered statistically significant. All analyses were performed using the Stata statistics program version 10.This study was performed in accordance with the Helsinki Declaration and was approved by the Ethics Committee of Stockholm (DNR 2014/2061–31 and 2017/129-32). All patients gave their oral and written consent.Plasma NT-proBNP was measured in 103 individuals of the 324 participants in the previous screening study. The analysis included 16 new cases of AF and all 34 known cases of AF. Altogether 53 (18%) individuals were selected from the 274 individuals with no detected AF and had their NT-proBNP levels measured. The clinical characteristics of those 53 individuals did not differ significantly from the remaining 221 individuals with no detected AF .New cases of AF had significantly higher NT-proBNP levels ) compared with individuals with no detected AF , p = 0.033). Moreover, cases with known AF had a significantly higher NT-proBNP level ) compared with individuals with newly detected AF (p = 0.026) and compared with individuals with no detected AF (p < 0.001) , Fig 2.The majority (68%) of cases with known AF had non-paroxysmal AF, compared to 37% of new cases. Of the 50 cases with AF in both known and new cases of AF, there were 29 cases of non-paroxysmal AF and 21 cases of paroxysmal AF. Cases with non-paroxysmal AF had a significantly higher NT-proBNP level ) compared to cases with paroxysmal AF ) (p < 0.001).In addition to the differences in the NT-proBNP levels among AF groups, the new cases of AF had greater prevalence of sleep apnea, higher diastolic blood pressure, and lower estimated glomerular filtration rate (eGFR) than cases with no detected AF. Cases with known AF had a greater prevalence of heart failure and history of stroke and higher CHA2DS2-VASc score than new cases of AF. Compared with cases with no detected AF, cases with known AF had a greater prevalence of heart failure and chronic obstructive pulmonary disease, higher CHA2DS2-VASc score, higher diastolic blood pressure, and lower eGFR . Women wIn the final multiple logistic regression model for the likelihood of detecting new AF, the NT-proBNP logarithmic mean and diastolic blood pressure were significantly associated with AF detection with an odds ratio and 95% CI of 2.29 (1.11 to 4.69) for log NT-proBNP and 1.08 (1.02 to 1.15) for diastolic blood pressure. In the final multiple logistic regression model for the association between known AF and new AF (the reference group), only heart failure was significantly associated with known AF with an odds ratio and 95% CI of 10.5 (1.24 to 89.92).Consequently, in the final multiple logistic regression model for the association between known AF and those with no detected AF (the reference group), heart failure, NT-proBNP logarithmic mean, and diastolic blood pressure were significantly associated with known AF with an odds ratio and 95% CI of 48 (3.7 to 622.7) for heart failure, 6.68 (2.66 to 16.77) for log NT-proBNP , and 1.1The AUC for discrimination of newly detected AF for NT-proBNP alone was 0.68 (95% CI 0.56 to 0.79) and for diastolic blood pressure alone was 0.71 (95% CI 0.60 to 0.83), but this difference was not statistically significant (p = 0.8). Adding diastolic blood pressure to NT-proBNP increased the AUC to 0.72 (95% CI 0.57 to 0.87). However, the differences in the AUC of the combined model versus either diastolic blood pressure alone or NT-proBNP alone were not statistically significant .To determine a cut-off level for NT-proBNP for AF screening, we considered at least 75% sensitivity as optimal in this non-acute setting. This resulted in a cut-off of 124 ng/L with a specificity of 45% and a negative predictive value of (24/28) 86%. Using this cut-off value, 28/69 (41%) of the patients would not have had to undergo screening by intermittent ECG. When Youden’s index was used instead to find the optimal cut-off for NT-proBNP, this resulted in a NT-proBNP cut-off value of 570 ng/L with a sensitivity of 31% and a specificity of 96%.Similar results using a cut-off diastolic blood pressure of 81 mmHg resulted in a sensitivity of 75% and a specificity of 53% with a negative predictive value of (33/37) 89%. Again, using a cut-off based on diastolic blood pressure, fewer individuals (32/69) 46% would need to go through AF screening with intermittent ECG recordings.This was the first screening study for AF in primary care where the role of NT-proBNP was evaluated. Participants in the screening program for AF who had newly detected AF had significantly higher NT-proBNP levels than individuals without AF. An NT-proBNP cut-off of 124 ng/L provided a reasonable balance between specificity and high sensitivity.There was low risk for selection bias among those who were selected from the screened population because all cases of new AF and known AF were included in the NT-proBNP assessment. Among the group of no detected AF, there were no statistically significant differences between those who were assessed for NT-proBNP and those who were not.A previous cohort study  showed tMoreover, NT-proBNP is an independent predictor for the development of cardioembolic stroke ,9, and tIn our study, the NT-proBNP level was higher 866 ng/L) in cases of non-paroxysmal AF compared to cases of paroxysmal AF (208 ng/L). These findings are in accordance with findings from a previous screening study [6 ng/L inThere were no differences between detected cases of AF and those with no AF regarding the CHA2DS2-VASc score and CHARGE-AF  score wiThe AUC for detecting AF was 0.68 for NT-proBNP in our study compared with 0.64 in the previous screening study [In our study, patients with known AF already detected by usual health care had more prevalent heart failure than new cases of AF detected by screening in the same age group. This difference might be attributed to the duration of AF. However, known AF cases had more non-paroxysmal AF than new AF cases. Progress of paroxysmal AF into persistent/permanent AF with increasing NT-proBNP is associated with structural cardiac remodeling and the progression to heart failure with associated comorbidities, including stroke. Therefore, early detection of AF by screening and early preventive measures might prevent or delay the development of heart failure and associated comorbidities.There are a few limitations to our study. The sample size was relatively small with low power to detect the associations between AF and many comorbidities. However, it could still detect the association between AF and NT-proBNP as well as the associations between AF and many comorbidities.Although the AF detection was adjusted for many possible confounders, there is still a need to adjust the AF detection for other unmeasured confounders such as echocardiogram variables.Using intermittent ECG recordings might underestimate the detection of AF. Intermittent ECG recordings detect a comparable proportion of AF as detection by five-day Holter monitoring. Prolonged Holter monitoring might detect a higher proportion of AF, but using intermittent ECG is easier and might be more acceptable by participants in the screening for AF than prolonged Holter monitoring.Finally, the NT-proBNP cut-off of 124 ng/L derived in this study has not been validated in the study; however, the previous study  showed aThe plasma level of NT-proBNP was significantly higher among primary care patients 70–74 years old with newly detected AF compared to those without AF, and therefore NT-proBNP might be used as a useful marker for AF detection. Higher levels of NT-proBNP were found in patients with non-paroxysmal compared with paroxysmal AF and in patients with known AF compared with new cases of AF; therefore, NT-proBNP might also be a marker for AF burden and associated comorbidities.S1 Table(XLSX)Click here for additional data file.


The aim of this study is to evaluate the microstructure and microcirculation of regional lymph nodes (LNs) in rectal cancer by using non-invasive intravoxel incoherent motion MRI (IVIM-MRI), and to distinguish metastatic from non-metastatic LNs by quantitative parameters.b = 0, 5, 10, 20, 30, 40, 60, 80, 100, 150, 200, 400, 600, 1000, 1500 and 2000 s/mm2) on a 3.0 T MRI system. One hundred sixty-eight regional LNs with a short-axis diameter equal to or greater than 5 mm from 116 patients were evaluated by two radiologists independently, including 78 malignant LNs and 90 benign LNs. The following parameters were assessed: the short-axis diameter (S), long-axis diameter (L), short- to long-axis diameter ratio (S/L), pure diffusion coefficient (D), pseudo-diffusion coefficient (D*), and perfusion factor (f). Intraclass correlation coefficients (ICCs) were calculated to assess the interobserver agreement between two readers. Receiver operating characteristic curves were applied for analyzing statistically significant parameters.All recruited patients underwent IVIM-MRI (D (P < 0.001), but lower f (P < 0.001) than the non-metastatic group. The area under the curve  of the multi-parameter combined equation for D, f and S was 0.811 . The diagnostic performance of the multi-parameter model was better than that of an individual parameter (P < 0.05).Interobserver agreement of IVIM-MRI parameters between two readers was excellent (ICCs> 0.75). The metastatic group exhibited higher S, L and IVIM-MRI parameters provided information about the microstructure and microcirculation of regional LNs in rectal cancer, also improved diagnostic performance in identifying metastatic LNs. The guidelines of the National Comprehensive Cancer Network (NCCN) emphasize the importance of neoadjuvant chemoradiotherapy for patients with rectal cancer . Nodal sKtrans) and fractional EES volume (ve) are helpful in distinguishing metastatic from non-metastatic LNs with sizes larger than 5 mm in rectal cancer [Dynamic contrast-enhanced MRI (DCE-MRI) allows both the qualitative and quantitative assessment of the tissue microcirculation , 6. Basel cancer . Howeverl cancer .Diffusion-weighted imaging (DWI) quantifies the Brownian motion of water molecules in both the intracellular and extracellular compartments without the need to inject any contrast agent . The appb-value DWI data [D), pseudo-diffusion coefficient (D*) and perfusion factor (f). These parameters allow inferences to be made about the microstructure and microcirculation of a biological voxel without the need to inject a contrast agent. The cutoff values and diagnostic accuracy of IVIM-MRI parameters in identifying nodal status in rectal cancer varies, as reported in a few previous studies [Intravoxel incoherent motion (IVIM) is the microscopic translational motion in each MRI voxel and includes the molecular diffusion of water and microcirculation of blood in the capillary network . IVIM-MRDWI data . The qua studies , 15, and studies , 15.In this prospective study, we sought potential information about the microstructure and microcirculation in LNs with S ≥ 5 mm from quantitative IVIM-MRI parameters and assessed whether these parameters are useful for predicting nodal status in rectal cancer.This study was approved by the medical ethics committee of the hospital, and written informed consent was obtained from each patient before participation.A total of 197 consecutive patients diagnosed with rectal carcinoma by endoscopic biopsy were recruited from January 2015 to August 2016. All patients with no history of pelvic surgery or contraindications to MR examination underwent preoperative MRI. Among these patients, 22 without scheduled curative surgery in our gastrointestinal department, as well as 50 receiving neoadjuvant chemoradiotherapy, were excluded. Nine patients were excluded because of poor MRI quality; the MR images of 6 patients had serious motion artefacts due to hip movements, and those of 3 patients had metal artefacts caused by titanium clips in the sigmoid colon or rectum. Thus, our prospective study enrolled 116 patients, 65 males and 51 females . The time between MR examination and surgery was a maximum of 2 weeks .A 3.0 Tesla MR system  with a 6-channel phased-array body coil was used for image acquisition. All patients were scanned in the supine position with a feet-first orientation. Rectal preparation was performed on the patients by pouring an appropriate amount (20–80 mL) of ultrasonic gel into the rectum, but this procedure was not performed if the tumours were large or located in the lower rectum. Unless contraindicated, 20 mg of raceanisodamine hydrochloride was injected intramuscularly 10–15 min before MR examination to reduce intestinal peristalsis and rectal spasm.b values  were applied to acquire the IVIM-MR images. In addition, fat-suppressed post-contrast T1WI and 3D-VIBE T1WI were acquired. The parameters for all the MRI protocols are shown in Table Pre-contrast axial, sagittal, coronal and oblique axial high-resolution turbo spin-echo T2-weighted images were obtained; the oblique axial planes were orthogonal to the tumour base. IVIM-MRI was performed in the axial orientation using a single-shot spin-echo echo-planar imaging pulse sequence prior to gadopentetate dimeglumine injection. A total of 16 Sb is the signal intensity at the particular b value we used, S0 is the signal intensity for b = 0 s/mm2, f is the perfusion factor representing the fractional perfusion linked to the microcirculation, D is the pure diffusion coefficient, and D∗ represents perfusion-related diffusion or incoherent microcirculation and is named the pseudo-diffusion coefficient.Based on the bi-exponential model introduced by Le Bihan et al. , the IVID, D* and f were generated automatically in a voxel-by-voxel manner using the former 14 b values. A segmented fitting algorithm was used for more robust parametric estimation [D and f were first estimated by linear least square fitting of the IVIM-MRI data with b ≥ 200 s/mm2, assuming that D* was significantly greater than D so that the influence of pseudo-diffusion on signal decay was negligible for b ≥ 200 s/mm2. Then, D* was estimated by applying the acquired D and f values to the equation with b < 200 s/mm2.Post-processing of the IVIM-MRI data was performed by using a prototype software-based MATLAB algorithm . The maps of timation . D and fb = 2000 s/mm2, a region of interest (ROI) was drawn manually on the cross-section of a node by referring to the corresponding T2W images, covering the nodal parenchyma as much as possible and excluding visible necrosis and vessels. The median D, D* and f of the ROI were estimated from the corresponding parametric maps [IVIM-MR images were post-processed independently by two experienced radiologists blinded to the histopathologic findings. First, we measured the morphological parameters, including the short-axis diameter (S), long-axis diameter (L) and the short-to-long axis diameter ratio (S/L), of each LN in the field of view (FOV) on the widest cross-section of the T2W images Fig. a. Then, ric maps . The entA node-for-node comparative scheme was constructed as follows by referring to previous articles , 18. A sAccording to the histopathologic findings, the radiologists re-matched the LNs with the preoperative IVIM-MR images in the corresponding groups, and a consensus was reached in cases of discrepancy between two readers. To ensure accurate node-for-node comparisons of the MR images and histopathologic findings, we devoted special attention to the nodal morphology in addition to the positions of the nodes relative to the tumour, rectal wall, mesorectal fascia, vessels, and adjacent nodes . The LNsStatistical analysis was performed using Statistical Package for the Social Sciences  and MedCalc Statistical Software .Interobserver agreement on each IVIM-MRI parameter for two independent readers was analysed by estimating the intraclass correlation coefficient (ICC). Based on the method given by Cicchetti , the ICCt test and the Mann-Whitney U test were applied for normally and non-normally distributed data, respectively.According to the results of the one-sample Shapiro-Wilk test, continuous data were expressed as the means ± standard deviations (SDs) or the medians with interquartile ranges (IQRs). The independent-samples Based on the results, we constructed receiver operation characteristic (ROC) curves for the statistically significant parameters derived from IVIM-MRI. Then, the optimum parameters were selected to establish a multi-parameter combined equation using the bi-logistic regression method. The ROC curve of predictive probability was also generated. The areas under the ROC curves (AUCs), with confidence intervals (CIs), were calculated. An AUC value of < 0.5 indicated no diagnostic performance; 0.5–0.7, poor performance; 0.7–0.9, moderate performance; and > 0.9, excellent performance. The maximum Youden indexes were used to determine the respective cutoff values producing the best diagnostic accuracy.P value of < 0.05 was considered statistically significant.The chi-square test or Fisher’s exact test was used to compare the clinical and pathological features of rectal tumours between node-negative and node-positive patients. A We harvested a total of 2089 LNs from the rectal specimens of 116 patients, with an average of 18 ± 10 nodes per patient. A total of 236 LNs from 49 patients contained metastases, and 1853 LNs were non-metastatic.In the node-for-node evaluations, 245 LNs found on IVIM-MR images were matched exactly with LNs found on histopathologic examination, whereas 1844 nodes  were not. Of the 245 matched LNs, 77 with a short-axis diameter of less than 5 mm were excluded from the evaluation. The remaining 168 LNs, including 78 malignant nodes and 90 benign nodes, were used for further analyses.The clinical features and pathologic findings of the primary tumours versus LN status in the 116 patients are shown in Table The ICCs of the IVIM-MRI parameters reflected excellent interobserver agreement between the two independent readers Table . The meaAccording to a previous study , we usedD values than the benign LNs (P < 0.001). However, a lower f was found for the malignant LNs than for the benign LNs (P < 0.001). S/L and D* were not significantly different between the two groups . The AUC (95% CI) of the ROC curve for logit P was 0.811 (0.744~0.868), which was greater than that of the other ROC curves when analysed via the pairwise comparison method (P < 0.05), thus demonstrating that the diagnostic performance of the combination of D, f and S was better than that of an individual parameter. Unfortunately, the AUCs of the ROC curves for the individual parameters were not significantly different (P > 0.05). The ROC curves and relative values are shown in Fig. The equation was indicated to be robust via the Hosmer-Lemeshow test (The AUC 9% CI of tD values of the metastatic LNs were higher than those of the non-metastatic LNs, while the f value was lower. Although the malignant LNs showed a lower D* than the benign LNs, we did not find a statistically significant difference between the two groups. The multi-parameter combined equation consisting of D, f and S improved diagnostic performance in defining nodal status.Our study showed that the IVIM-MRI parameters exhibited excellent interobserver agreement between two independent readers, consistent with the results of a previous study . The gooGenerally, tumour cell proliferation induces nodal enlargement. Although some published studies used size criteria to detect nodal metastasis in rectal cancer, the cutoff value for the short-axis diameter lacked consistency –4. A patD* and f parameters derived from IVIM-MRI are related to the microcirculation [f represents a volume fraction of water flowing in perfused capillaries, which mainly depends on the density of active capillaries [f, indicating a decrease in the density of active capillaries in positive nodes. We explained this result as follows. The blood supply of a LN is provided by one or more arterioles, which then branch into the capillary network [f decreases in metastatic LNs, as we found. Our result was consistent with that of Yu et al. [f than non-metastatic LNs in cervical cancer [f is mainly affected by relaxation effects and the T2 contribution [b value distribution also influence the estimation of this parameter [According to the bi-exponential equation given by Le Bihan et al., both the culation . The perillaries . We foun network , 23. Cap network . Neoangi network . Instead network . Accordiu et al. . In addil cancer . Howeverl cancer  contrastribution , 27, thearameter , 28.D* is proportional to the mean capillary segment length and blood velocity, reflecting the blood volume flowing inside microvessels [D* values than benign LNs, which is corroborated by two of the abovementioned studies [D* values in malignant LNs than in non-malignant LNs, but we did not find a statistically significant difference between the two groups, a result similar to that of the study by Wu et al. [D* values than negative LNs [b values [b values lower than 200 s/mm2 improves the characterization of the pseudo-diffusion effect [b values of less than 200 s/mm2, D* exhibited the lowest ICC of the IVIM-MRI parameters, but its utility was still unclear. The utility of D* might be limited by its high uncertainty and poor reproducibility, as previously reported [Based on the IVIM theory, the pseudo-diffusion coefficient ovessels . Becauseovessels , metasta studies , 15. Ouru et al. . This lative LNs , 29. In b values ; the incn effect , 31. Altreported –35.D is the pure diffusion coefficient, which is related to tissue microstructure; it reflects the molecular diffusion of water in extracellular and intracellular spaces and the exchange between these two compartments [D is inversely correlated with the cellularity and the cellular nucleus-to-cytoplasm ratio [D of metastatic LNs found in our study, which was concordant with the results of the study by Qiu et al. [D [D of metastatic LNs in breast cancer [artments . Generalsm ratio . Becausesm ratio . Howeveru et al. . On the t al. [D . The sint al. [D . Our whot al. [D . Varioust cancer , lung cat cancer  and headt cancer . The inct cancer .D, f and S showed moderate diagnostic performance in distinguishing metastatic from non-metastatic LNs and performed better than the individual parameters. Thus, we believe that IVIM-MRI parameters have potential value in defining nodal status because they can provide information about the microstructure and microcirculation. Based on the results, contrast-enhanced MRI using gadolinium should not be mandatory for every rectal cancer patient.The multi-parameter model consisting of b values lower than 200 s/mm2 requires further study.Some limitations of our study need to be carefully considered. First, we excluded LNs with a short-axis diameter of less than 5 mm because the smaller number of pixels makes these LNs susceptible to contamination by misregistration effects or partial volume effects from adjacent tissues. However, approximately 50% of malignant LNs in rectal cancer are smaller than 5 mm in diameter. Second, we did not analyse different histopathologic types of metastatic LNs in rectal cancer, for example, mucinous versus nonmucinous carcinoma. Third, we did not evaluate the effects of neoadjuvant chemoradiotherapy on the diffusion and perfusion parameters of LNs. Fourth, the distribution of D and f values derived from IVIM-MRI differed significantly between metastatic and non-metastatic LNs in rectal cancer. The diffusion and perfusion parameters provided additional information about the microstructure and microcirculation. Thus, the multi-parameter model comprising D, f and S exhibited improved performance in diagnosing nodal status.In conclusion, the


At the sampling rate fS = 9 kS/s, the proposed ADC achieves a signal-to-noise and distortion ratio (SNDR) of 55.5 dB and a spurious-free dynamic range (SFDR) of 70.6 dB. The proposed dual calibration technique improves the SFDR by 12.7 dB. Consuming 1.15 μW at fS = 200 kS/s, the ADC achieves an SNDR of 55.9 dB and an SFDR of 60.3 dB with a figure-of-merit of 11.4 fJ/conversion-step.Herein, we present an energy efficient successive-approximation-register (SAR) analog-to-digital converter (ADC) featuring on-chip dual calibration and various accuracy-enhancement techniques. The dual calibration technique is realized in an energy and area-efficient manner for comparator offset calibration (COC) and digital-to-analog converter (DAC) capacitor mismatch calibration. The calibration of common-mode (CM) dependent comparator offset is performed without using separate circuit blocks by reusing the DAC for generating calibration signals. The calibration of the DAC mismatch is efficiently performed by reusing the comparator for delay-based mismatch detection. For accuracy enhancement, we propose new circuit techniques for a comparator, a sampling switch, and a DAC capacitor. An improved dynamic latched comparator is proposed with kick-back suppression and CM dependent offset calibration. An accuracy-enhanced bootstrap sampling switch suppresses the leakage-induced error <180 μV and the sampling error <150 μV. The energy-efficient monotonic switching technique is effectively combined with thermometer coding, which reduces the settling error in the DAC. The ADC is realized using a 0.18 μm complementary metal–oxide–semiconductor (CMOS) process in an area of 0.28 mm Demand is increasing for various battery-operated sensing systems, for example, the Internet of Things (IoT), which is deployed in various objects for biomedical, home, industrial, and environmental monitoring . For theTo meet the demand, the successive approximation register (SAR) analog-to-digital converter (ADC) has drawn much interest, due to its medium conversion rate and low-power consumption. Various approaches have been proposed to further reduce power consumption. Compared with the conventional structure, the average energy can be reduced by 37.5% using a capacitor splitting technique . When moMonotonic switching uses energy-efficient down switching only. The switching is efficiently realized using a reduced number of switches and capacitors, which simplifies the design of SAR logic . With doMismatch in the capacitive DAC, which occurs due to process variations and routing parasitics, limits the linearity of the ADC. Several techniques have been reported to calibrate the mismatch ,8,9. In Taking advantage of scaled-down complementary metal–oxide–semiconductor (CMOS) process, various digital calibration techniques have been reported ,11,12,13In this paper, we present a low-power SAR ADC realized in an energy and area-efficient manner for a sensor interface. The proposed ADC features (1) fully-integrated on-chip dual calibration and (2) various accuracy-enhancement techniques.(1)Dual calibration: The proposed calibration techniques, CM-dependent comparator offset calibration (COC) and DAC capacitor mismatch calibration, are realized in an energy and area-efficient manner. By reusing the DAC for generating calibration signals, the CM-dependent comparator offset is calibrated without using separate circuit blocks for calibration. After COC, DAC capacitor calibration is efficiently performed by reusing the comparator for delay-based mismatch detection.(2)Accuracy-enhancement techniques: To support the calibration operation, we propose new accuracy-enhancement techniques for a comparator, a sampling switch, and a DAC capacitor. We present a dynamic latched comparator, which is robust to kick-back noise. An improved bootstrap sampling switch is proposed, which suppresses a leakage-induced error within 180 μV and a sampling error less than 150 μV. The monotonic switching technique is effectively combined with thermometer coding to reduce the settling error in the DAC.The measured data indicate the successful operation of the ADC and performance improvement by the proposed dual calibration technique. At a sampling rate of 200 kS/s, the ADC achieves a signal-to-noise and distortion ratio (SNDR) of 55.9 dB and a spurious-free dynamic range (SFDR) of 60.3 dB with a figure-of-merit (FoM) of 11.4 fJ/conversion-step.T[i] and binary-weighted capacitors CB[i] (i = 0 to 6). Thermometer coding reduces the size of the most significant bit (MSB) capacitor from 256 to 64 unit capacitors. Thus, the settling error in the DAC can be reduced.A straightforward way to reduce the settling error is reserving different delay times for each DAC capacitor . This apT[i] arrays. To realize the calibration in an energy and area-efficient manner, the lower CB[i] are not calibrated; they require sufficient intrinsic linearity. Otherwise, they set the upper limit on performance.The proposed ADC supports a dual calibration technique for COC and DAC capacitor mismatch calibration. During the COC, we note that the capacitive DAC is not used for analog-to-digital (A/D) conversion. To realize the calibration without using separate circuit blocks, the DAC is reused for implementing the common-mode voltage generators (CMVG). During COC, a multiplexer (MUX) array selects inputs from the CMVG for the DAC. During normal A/D conversion, the inputs from SAR logic are selected. The linearity of SAR ADC is affected by the mismatch in the capacitive DAC. Because high mismatch errors occur for the large capacitors, the DAC mismatch calibration is applied to upper CVoffset; (3) during monotonic switching, Voffset depends on the CM voltage VCM.The dynamic latched comparator is widely used to reduce power consumption . We consVDACP,N which are disturbed by the clock transition. In the dynamic latched comparator, the input difference is resolved when the clock signal CLK is switched from low to high. By the CLK transition, the kick-back noise is generated at the input of the comparator by clock feed-through . This operation repeats until all nine VCM steps are processed. During normal A/D conversion, the stored data in the register are sequentially read using MEM_OUT[4:0], which sets the state of the capacitor array.With the same VCM is gradually reduced, the CMVG generates nine VCM steps of each 112.5 mV in the range from 0.9 to 0 V. Each VCM is generated by controlling the bottom plate of the capacitor arrays CT[6:0] and CB[6:0]. With a reference voltage of 1.8 V, a VCM step of 112.5 mV corresponds to 32 CU (CU = 31.7 fF is a unit capacitor). To complete one cycle of COC, eight clocks are needed; one clock for reset, five clocks for determining the state of the capacitor array, and two clocks for the data store. Therefore, we use a CLK/8 divider for the CMVG. The VDACP,N changes its value at every rising edge of CLK/8.VCM step, the rising edge of CAL_WR is used for reset. During the period when CAL_WR = 1, the CMVG generates a VCM to determine the state of the capacitor array. Then, the calibration data MEM_IN[4:0] are stored with the MEM_EN signal. When offset measurement for nine VCM is finished, CMP_CAL_EN signal becomes low, which indicates the end of comparator offset measurement.VCM is already close to the ground and does not change significantly. Therefore, Cal_1[4:0] is used during this period. With the end-of-conversion (EOC), the ADC generates outputs.T[i] and CB[i]. We note that the one-bit of CT[i] has a weight of 64CU. Under the ideal matching condition, the sum of binary capacitors from CB[6] to CB[0] has the same weight, 64CU. For the DAC capacitor mismatch calibration, we detect the difference between VDACP and VDACN, which can be used to evaluate the mismatch between the upper and lower DAC. The proposed approach is different from the previous works . Due to circuit complexity, CB[i] is not calibrated.us works ,8,11, whT[i] is sequentially detected by using the sum of CB[i] in the other branch. The positive DAC branch is evaluated first and the negative DAC branch is calibrated next. The procedure can be summarized as follows:(1)Before starting mismatch calibration, the bottom nodes of all capacitors in the DAC are reset by connecting them to the ground.(2)T[0], connect the bottom plate of CT[0] in the positive branch to the reference voltage VREF and generate VDACP. Then, connect the bottom plate of all CB[6:0] in the negative branch to VREF and generate VDACN. If there is a mismatch, the difference between VDACP and VDACN is reflected as the output delay in the comparator.To evaluate the mismatch error of C(3)T[0] in the positive branch.The delay in the comparator is encoded using two-bit data LSB_P[1:0], which represents mismatch information for C(4)T[1] − CT[6]) in the positive DAC branch.Sequentially evaluate the mismatch of the remaining thermometer-coded capacitors (C(5)T[i] in the negative DAC branch. This mismatch information is encoded using two-bit data LSB_N[1:0].In the same manner, evaluate the mismatch of seven CUsing the symmetric properties of the differential structure, the proposed calibration method measures the mismatch between the upper and lower DAC. The mismatch in the one-bit CT[0:6], the mismatch data are retrieved from the register. There are seven register outputs for positive  and negative  branches. These outputs are used to set the compensation capacitors attached to each CT[0:6].T[0] in the positive branch. During this time, LSB_N_CNTL controls the bottom plate of capacitors CB[6:0] in the negative branch. The LSB_N_CNTL is enabled until mismatch evaluations are performed for all CT[0:6] in the positive DAC branch. The negative branch calibration logic operates in a similar manner using LSB_P_CNTL. When the DAC mismatch calibration is finished, CAL_END becomes low.T[0] in the positive branch is evaluated  is independent of ΔVin but affected by the process corner. The second term is inversely proportional to ΔVin.The comparator delay depends on the DAC capacitor mismatch. When the mismatch error of Cated See , for exaobtained  using:(2e tlatch  as:(3)tDM7,8 See b, IBIAS2Vin of 0.6 and 0.9 LSB for 1.0% and 1.5% capacitor mismatch. Considering some margin for the mismatch, the delay generator is sized to produce proper delay so that mismatch error from 0.5 to 1.5 LSB is detected. To prevent malfunction, we determine the proper delay in the comparator and the delay generator by performing extensive Monte-Carlo simulations. Because the delay in these circuits shares a global process corner, we are able to mitigate the mismatch between delay detectors by using careful layout.Using (2), we obtain ΔVin. Therefore, the delay threshold for PSET_D1,D2 is chosen by using three equal delay regions. To deal with the process variation, the delay is further tuned using VTUNE1,2 in the delay generator.VDACP and VDACN is also small, resulting in a relatively long delay in the comparator [mparator . For exaT[0:6] in the two branches are generated. By the calibration logic, the data are sequentially written in the register  of 64CU. The effect of calibration can be enhanced by using both add and subtract operations. The subtract operation is implemented by taking advantage of the differential structure  and LSB_N[1:0]) for each Cregister . The dattructure . To simpIn order to assess the performance improvement by the proposed calibration technique, behavioral simulations are performed using Matlab. By including random DAC capacitor mismatch in the behavioral model of the ADC, we perform 1000 Monte-Carlo simulations. Comparator and kT/C noises are not included. The foundry datasheet shows 1% capacitor mismatch which is a conservative estimate. Because there is additional mismatch caused by routing and fringing components, we consider the random mismatch from 1.0% to 2.5%.VDACP and VINP occurs due to the on-resistance of the switch. To reduce the resistance, a boosted voltage of about 2VDD is applied to the gate of N1 and N2 through the capacitor CC2. In addition, P1 in parallel with N1 forms a transmission-gate which further reduces the on-resistance. To reduce the leakage-induced error, the threshold voltages of N1 and N2 (both are inside deep n-well) are increased by controlling the body terminal. When the sampling clock CLKS is high, the body voltage Vb1,2 is set to the threshold voltage of P2 by the clamping circuit. When CLKS goes low, Vb1,2 is reduced by VDD through the capacitor CC1, which is about −1.3 V. The size of these transistors is optimized by considering the tradeoff between the on-resistance and leakage-induced error.The sampling error defined by the difference between VINP. The result is obtained from post-layout simulations with a sampling rate of 3 kS/s. The VDACP is measured at 1 ms after the input VINP is sampled. Three cases of the bootstrap switch (BS) are shown; (1) BS-1 having transmission-gate without a clamping circuit; (2) BS-2 having a clamping circuit and the series switch (N1 and N2) only [VINP reaching 800 μV at VINP = 1.8 V. The result shows that BS-3 has a smaller error than that of BS-2 except at VINP = 1.8 V; BS-3 shows a leakage-induced error less than 50 μV up to VINP = 1.7 V. The worst-case error is 180 μV. VINP. The turn-on voltage of the sampling switch is 2VDD. The gate-to-source voltage Vgs of the switch, thus, the turn-on resistance depends on VINP. This causes the sampling error to vary with the input. A constant Vgs bootstrapping technique is reported [N2) only ; (3) BS-reported ; it can reported ; this ap2.fIN. fS up to 100 kS/s. We characterize the ADC using a high fIN.fIN = 20.35 kHz. When both COC and DAC calibration are performed, the measured SNDR and SFDR are 56.2 and 70.3 dB, respectively, resulting in an ENOB of 9.0 bits. At the Nyquist frequency, the measured SNDR and SFDR are 55.9 and 60.3 dB, respectively.fIN. The SFDR decreases with fIN and the SNDR remains relatively constant up to 100 kHz. fS. Both SNDR and SFDR remain relatively constant up to fS = 200 kS/s.The overall power consumption of the ADC is 1.15 μW. The power breakdown shows that the DAC, the comparator, SAR logic, and calibration blocks consume 101 (8.8%), 516 (44.9%), 280 (24.3%), and 253 nW (22%), respectively. Comparison with the other works is shown in 2 using 0.13 μm CMOS. Our work is realized using 0.18 μm CMOS in a compact chip area of 0.28 mm2. Both the works [fS = 20 and 1 kS/s, respectively. To capture all these tradeoffs, we can use the figure-of-merit (FoM), which is defined as:fS = 200 kS/s, the ADC in this work achieves a good FoM of 11.4 fJ/conv-step.The work in reference  presentshe works  show rel work in  shows a fS = 9 kS/s, the proposed ADC achieves the measured SNDR of 55.5 dB and the SFDR of 70.6 dB. At an increased fS = 200 kS/s, the ADC achieves an SNDR of 55.9 dB and an SFDR of 60.3 dB with a FoM of 11.4 fJ/conversion-step. The results indicate the potential of our work for low-power sensing applications.We present a low-power SAR ADC with dual on-chip calibration technique applied for COC and DAC capacitor mismatch correction. The proposed calibration technique is realized in an energy and area-efficient method that does not use separate circuit blocks. In addition, various accuracy-enhancement techniques further improve the performance of the ADC. A monotonic switching technique is efficiently combined with thermometer coding to reduce the error caused by incomplete settling. The CM-dependent comparator offset is dynamically calibrated by reusing the differential DAC. In addition, the dynamic latched comparator is carefully designed to remove decision errors due to kick-back noise. The evaluation of the DAC capacitor mismatch is performed by reusing the comparator for delay measurement. The calibration of the DAC mismatch in the thermometer-coded seven MSB bits is efficiently performed by using the symmetry property of the differential structure. Measured data show the successful operation of the proposed dual calibration technique. At